Methods of inducing immunity using low molecular weight immune stimulants

ABSTRACT

The present invention relates to peptide-like compounds, eg aminocarboxylic acid amide derivatives, and to methods of using same to stimulate cells of the immune system, bone marrow and other organs. The present compounds can be used to enhance vaccination, increase synthesis of and enhance function of blood cell components and enhance anti-neoplastic effects of various agents. The compounds of the invention can be used to produce a variety of further pharmacologic effects.

This application is based on and claims priority from ProvisionalApplication No. 60/005,336, filed Oct. 17, 1995.

TECHNICAL FIELD

The present invention relates to peptide-like compounds, egaminocarboxylic acid amide derivatives, and to methods of using same tostimulate cells of the immune system, bone marrow and other organs. Thepresent compounds can be used to enhance vaccination, increase synthesisof and enhance function of blood cell components and enhanceanti-neoplastic effects of various agents. The compounds of theinvention can be used to produce a variety of further pharmacologiceffects.

BACKGROUND

A variety of polypeptide cytokines, hormones and immune systemmodulators have been used to stimulate production and activity of bonemarrow-derived cells. However, little progress has been made inobtaining the same physiologic activities in culture and in vivo usingsimple, chemically synthesized small molecules. For example, there arerelatively few reports of the use of simple, small molecules instimulating production and function of various blood components,including, without limitation, red blood cells (RBCs) and white bloodcells (WBCs), in stimulating the response to vaccinations, in enhancingdifferentiation and in the nontoxic treatment of neoplasia. The presentinvention relates to such methods, as well as others, and to compoundssuitable for use in same.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide compounds that exertan immunomodulatory effect. It is another object of the invention toprovide a method of altering (eg stimulating) cellular productivity andvitality and to provide a method of modulating cell growth.

It is a specific object of the invention to provide a method ofmodulating immune function, for example, to facilitate vaccinationagainst or treatment of diseases, including infectious and autoimmunediseases, as well as other diseases in which the immune system plays arole.

It is another specific object of the invention to provide a method ofeffecting blood cell stimulation (including RBCs, WBCs, stem cells,platelets and others).

It is a further specific object of the invention to provide a method ofenhancing cell differentiation and cell growth and a method of exertingan anti-senescence effect in vitro and in vivo. It is also a specificobject of the invention to provide a method of preserving viability ofneurons, natural killer (NK) cells, fibroblasts and other cell types invivo and in vitro. It is a further specific object of the invention toprovide a method of exerting anti-Alzheimer and anti-aging effects and amethod of treating genetic diseases related to aging. It is a furtherobject of the invention to provide a method of enhancing bioactivity ofcosmetics and compounds to serve as cosmeceuticals.

It is also a specific object of the invention to provide a method oftreating a neoplastic or preneoplastic condition. It is another objectof the invention to provide a method of ameliorating side effects ofvarious anti-neoplastic agents.

It is a further specific object of the invention to provide a method ofaltering (eg stimulating) cellular protein production, includingantibody production.

The foregoing objects are met by the present invention which providesaminocarboxylic acid amide derivatives that can be used to produce avariety of biomodulatory effects, both in vivo and in vitro.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows synthetic reaction schemes.

FIG. 2A-D show specific compounds of the invention bearing carbondesignations.

FIG. 3 shows specific compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the present invention relates to compounds of theformula (I): ##STR1## wherein: A is a group of the formula --PO₃ H,--SO₃ H, --OPO--(OH)₂, --OSO₂ OH, or --SH, or pharmaceuticallyacceptable salt thereof or physiologically hydrolyzable derivativethereof, or disulfide thereof when A is --SH. Suitable salts includesodium, potassium, calcium and zinc. Suitable hydrolyzable derivatizinggroups include esters, such as substituted or unsubstituted lower alkyl(eg C₁ to C₄) or arylalkyl (eg benzyl) esters;

R₁ is H, a linear or branched lower alkyl, for example, a C₁ to C₆alkyl, arylalkyl, for example, wherein the alkyl moiety is C₁ to C₄alkyl and the aryl moiety is a substituted (eg lower alkyl or halogen)or unsubstituted phenyl group, or alkenyl (for example, C₂ -C₆ alkenyl);

R₂ is H, a linear or branched lower alkyl, for example, a C₁ to C₆alkyl, an alkenyl, for example, a C₂ -C₆ alkenyl, an arylalkyl, forexample, wherein the alkyl moiety is a C₁ to C₄ alkyl and the arylmoiety is a substituted (eg lower alkyl or halogen) or unsubstitutedphenyl group; or an acyl, for example, acetyl, benzoyl, arylsulfonyl(for example, when the aryl moiety is phenyl); a carbonate ester such asalkoxycarbonyl (eg, C₁ -C₇ alkoxy carbonyl) (for example, --OCOC(CH₃)₃);allyloxy carbonyl (eg --OCOCH₂ CH═CH₂); cycloalkoxycarbonyl (eg when thering is C₃ -C₈ (C₅ -C₆ being preferred) and when the alkoxy moiety is C₁-C₈) (for example --OCOCH₂ C₅ H₉); or an unsubstitutedarylalkoxycarbonyl (for example --OCOCH₂ C₆ H₅) or a substitutedarylalkoxycarbonyl wherein the substituent is, for example, a halogen, anitro group, an amino group or a methoxyl group;

alternatively, R¹ and R² taken together form, with the nitrogen to whichthey are attached, a 5 to 7 membered ring (for example, R¹ and R² takentogether can be --(CH₂)₄ --, --(CH₂)₅ or --(CH₂)₆ --); and

L¹ and L² are hydrocarbon linking groups, for example, a linear orbranched chain alkyl of the formula --(C_(n) H_(2n))-- wherein n is, forexample, 1 to 8 in the case of L¹ and 2 to 8 in the case of L² exceptwhen A is --PO₃ H or --SO₃ H in which case n can be 1-8; a cycloalkyl of3 to 8 carbon atoms, preferably 5 or 6 carbon atoms; or aninterphenylenel ##STR2##

Advantageously, L¹ and L² are --(C_(n) H_(2n))-- wherein n is 1 to 8 inthe case of L¹ or 2 to 8 in the case of L² except when A is --PO₃ H or--SO₃ H in which case n can be 1-8 (examples of branched chain alkylsinclude --CH₂ CHR--, --CH₂ CHRCH₂ --, --CHRCH₂ CH₂ --, and --CH₂ CH₂CHR-- wherein R is an alkyl group and wherein the total number of carbonatoms, including R, does not exceed 8).

A particular group of compounds of the invention is of the formula (I)wherein A, R¹, R², L¹ and L² are as defined above in said firstembodiment with the proviso that when A is --SO₃ H or pharmaceuticallyacceptable salt thereof or physiologically hydrolyzable derivativethereof, one of R¹ and R² is H, and L¹ and L² are (CH₂)₂, then the otherof R¹ and R² is not H.

Another particular group of compounds of the invention is of the formula(I) wherein A, R¹, R², L¹ and L², are as defined above in the firstembodiment with the proviso that when A is --SO₃ H or pharmaceuticallyacceptable salt thereof or physiologically hydrolyzable derivativethereof, one of R¹ and R² is H, and L¹ and L² are (CH₂)₂, then the otherof R¹ and R² is not C₆ H₅ CH₂ OCO--.

A further particular group of compounds of the invention is of theformula (I) wherein A is a group of the formula --PO₃ H or --OPO(OH)₂,more particularly --PO₃ H, or a pharmaceutically acceptable salt thereofor a physiologically hydrolyzable derivative thereof, and wherein R¹,R², L¹, and L² are as defined above in the first embodiment.

Another particular group of compounds of the invention is of the formula(I) wherein A is a group of the formula --SO₃ H or --OSO₂ OH, moreparticularly --OSO₂ OH, or pharmaceutically acceptable salt thereof, orphysiologically hydrolyzable derivative thereof, and wherein R¹, R², L¹and L² are as defined above in the first embodiment. The provisos abovecan apply to this group of compounds as well.

A further particular group of compounds of the invention is of theformula (I) wherein at least one of R¹ and R² is an alkyl,advantageously a lower alkyl (eg C₁ to C₆), and wherein A, L¹, L² andthe other of R¹ and R² are as defined above in the first embodiment.

Another particular group of compounds of the invention is of the formula(I) wherein R¹ is an alkyl and R² is acyl and wherein A, L¹ and L² areas defined above in the first embodiment.

A further particular group of compounds of the invention is of theformula (I) wherein L¹ is --(CH₂)-- and wherein A, R¹, R², and L² are asdefined above in the first embodiment.

Yet another particular group of compounds of the invention is of theformula (I) wherein R¹ and R² are taken together and form, with thenitrogen to which they are attached, a 5 to 7 membered ring, and whereinA, L¹ and L² are as defined above in the first embodiment.

The present compounds can also be present covalently bound to proteins,for example, antigens or other immunologically active proteins, or celltargeting proteins. Such conjugates can be synthesized using techniquesknown in the art.

The compounds of the present invention can be prepared using, forinstance, methods provided in the Examples and in U.S. Pat. No.4,102,948 and U.S. Pat. No. 4,218,404, as appropriate.

In another embodiment, the present invention relates to methods of usingthe above-described compounds in vivo and in vitro to alter (egincrease) cellular productivity and vitality and to modulate cellulardifferentiation, growth and/or function.

In vivo, the compounds can be used to elicit a variety of responses,including stimulating bone marrow and platelet production, stimulatingerythropoiesis, altering (eg increasing) immunogenic responsiveness andtreating neoplasia. For example, the present compounds can be used totreat anemia and neutropenia. The compounds of the invention can be usedto treat or prevent premature aging and degenerative diseases and totreat inherited metabolic diseases. The compounds of the invention canbe used in the treatment of diseases of immune dysfunction including,without limitation, autoimmune diseases such as rheumatoid arthritis,diabetes, thyroiditis, lupus (SLE), connective tissue diseases, multiplesclerosis, sarcoidosis, psoriasis, hepatitis, and kidney diseases. Thecompounds can be used, for example, in the treatment of genetic diseasesof aging (Ataxia telangiectasia, progeria and Werner's syndrome), inaccelerated aging (as compared to the ultimate biologic potential of theorganism), and in the treatment of Alzheimer's disease. The presentcompounds can be used to delay sensecence of fibroblasts, neural,lymphoid, epithelial, endothelial, mesenchymal, neuroectoderm,mesothelial and other cells, and to maintain function and health of agedcells and organisms.

The compounds can be used to cause an alteration in the number of cellsof a particular cell type (eg epithelial cells or mesenchymal cells)(the compounds can be used, for example, to increase the number of redcells or white cells or the numbers of neuronal cells) or to cause analteration in cellular function (eg an increase phagocytic activity ofmacrophage).

From the standpoint of immunogenic responsiveness, the present compoundscan be used to enhance antigen processing, cell to cell communication,cellular immunity, natural immunity, humoral immunity, macrophagefunction, NK cell function, immune surveillence, immune response andimmune killing. Further, the compounds of the invention can be used inconjunction with vaccination protocols to alter (eg increase) theresponse elicited by an antigen or an immunogenic conjugate. The presentcompounds can be used in vaccinations against infectious, neoplastic,autoimmune and other diseases. Specifically, the present invention canbe used to enhance vaccinations to bacterial and viral diseases, forexample, pneumonia, meningitis, TB, hepatitis B and HIV and to parasiticdiseases. Further examples include such bacteria and bacterial diseases:Pyogenic cocci (staphylococci, pharyngitis, tonsillitis, sinusitis,streptococci, pneumococci, meningococci, gonococci), enteric bacilli(Escherichia coli, Klebsiella, Salmonella shigella), cholera,pseudomonas (Pseudomonas aeruginosa, Pseudomonas mallei), bacteroides,mycobacteria (tuberculosis), spirochetes (Treponema pallidum(syphilis)), clostridia, diphtheria Hemophilus and Bordetella bacilli,Granuloma inguinale, brucella, tularemia, anthrax, plague, mycoplasma,listeriosis; rickettsial disorders: typhus group, Rocky Mountain spottedfever, Lyme disease, scrub typhus, Q fever; chlamydial disorders:trachoma and inclusion conjunctivitis, lymphogranuloma venereum, andpsittacosis; viral diseases: cutaneous viral infections (chickenpox,herpes zoster, measles), respiratory viral infections, viral diseases ofthe central nervous system, viral diseases of the liver, viral diseasesof the salivary glands, and infectious mononucleosis; fungal diseases:candida albicans, mucor, histoplasmosis, aspergillosis, blastomycosis,coccidioidomycosis, actinomycosis and nocardiosis; and protozoal(parasitic) diseases: pneumocystosis, amebiasis, malaria, toxoplasmosis,leishmaniasis, trypanosomiasis, and giardiasis; helminths diseases(worms): trichinosis, strongyloidiasis, enterobius vermicularis,filariasis, hookworm disease, ascariasis, flukes, cestodes, tapeworms,and trichuriasis; and other diseases: sarcoidosis, cat-scratch disease,legionnaires' disease.

The compounds of the invention can also be used to inhibit the toxicityassociated with immunotoxic and carcinogenic agents.

Depending on the effect sought and the clinical situation, the compoundsof the invention can be administered before, during or aftervaccination. Use of the present compounds can result in more effectiveinjections and/or a reduction in the number of injections necessary forvaccination. The present compounds can also be used to treat infections,including chronic infections.

In a specific embodiment, the invention relates to a method of effectingisotype conversion using the compounds of the present invention. Asshown in Example IX, the present compounds can be used to effect rapidinduction of immunoglobulin G. These data demonstrate that the presentcompounds can be used to elicit a rapid response to a vaccine therebyreducing the number of injections necessary and/or increasing theefficiency of the each injection. The compounds of the invention canalso focus antibody production of the polysaccharide and thus effectexcellent responses to polysaccharide antigens, whether or notconjugated to a protein carrier.

As to neoplasia treatment, the compounds of the invention can be used totreat a variety of preneoplastic and neoplastic conditions, includingboth soft (eg hematolymphoid) and solid tumors (eg carcinomas andsarcomas). More specifically, the compounds of the invention can be usedto treat breast cancer, prostate cancer, glioblastomas, melanomas,myelomas, lymphomas, leukemias, lung cancer, skin cancer, bladdercancer, kidney cancer, brain cancer, ovarian cancer, pancreatic concer,uterine cancer, bone cancer, colorectal cancer, cervical cancer andneuroectodermal cancer, and premalignant conditions, including, withoutlimitation, monoclonal gammapothies, dysplasia, including, withoutlimitation, cervical and oral dysplasia. The compounds can also be usedto treat conditions associated with altered differentiation (eg loss ofpigmentation, hair; alteration of skin including psoriasis; alterationof gastrointestinal, kidney, liver, brain, endocrine, immune, lung,connective tissue, cardiac or other organ function).

The compounds of the invention can be administered topically, orally,rectally, intravaginally intravenously, intraperitoneally,subcutaneously, intramuscularly or intranasally, as appropriate for theeffect sought. The compounds can also be administered transdermallyusing, for example, transdermal patches or transmucosally via sprays orother application.

Dosages of the present compounds can range, for example, fromfemtograms/kg body weight to nanograms/kg body weight in the case ofdisease prevention or function/health improvement to about 10 μg/kg b.w.to about 100 mg/kg b.w. in disease treatment regimens. Optimum doses forany particular situation can be determined by one skilled in the artwithout undue experimentation, often using appropriate in vitro oranimal models.

The compounds of the invention are typically used in the form of apharmaceutical composition comprising the compound of formula I, or saltor hydrolyzable derivative thereof as described above, together with apharmaceutically acceptable diluent or carrier. The composition can bepresent in dosage unit form, for example, as a tablet, capsule orsuppository. The composition can be formulated so as to be suitable fortopical application (eg as a gel, cream, lotion or ointment).Alternatively, the composition can be present as a solution orsuspension (eg sterile) suitable for administration by injection,inhalation, intranasally or dropwise to the eye or other sites asappropriate. The compound of the invention can be prepared as a slowrelease formulation appropriate for internal or external use. Usingtechniques known in the art, the compounds of the invention can also betrapped in or bound to a polymer, a solid support, a liposome or a gel.

Carriers and diluents known in the art can be used and the composition,when, for example, in the form of a tablet or capsule, can be formedwith an enteric coating.

The composition of the invention can include active agents in additionto the compounds of formula I. Examples of such additional active agentsinclude cancer chemotherapeutic agents, hormones, vitamins, cytokines,enzyme regulators, regulatory macromolecules, regulatory thiols or othersmall molecules.

The present compounds also have ex vivo applications including in thegrowth, maintenance or differentiation of tissue grafts, including boneand vascular grafts, and in the treatment of cells and organs, forexample, prior to transplantation or use in the laboratory.

While the compounds of the invention are suitable for therapeutic use inhumans, the compounds of formula I are also useful in the veterinarytreatment of similar conditions affecting warm-blooded animals, such asdogs, cats, horses and cattle. For such purposes, the compounds of theformula I can be administered in an analogous amount and manner to thosedescribed above for administration to humans. The compounds of theinvention also have application to lower organisms, including insects,reptiles, birds, fish and plankton, microorganisms, or others. They canbe used in aqueous environments, including in marine or fresh watersettings. For example, the present compounds can be used to alter (egstimulate) fish growth, development and/or reproduction, for example, inaqua cultures.

In addition to the usefulness of the present compounds in connectionwith intact animals (particularly, but not exclusively, mammals) cells,tissues and organs, the compounds of the invention also have applicationin agricultural settings to alter (eg stimulate) plant growth anddevelopment as well as plant cell function and productivity. Thecompounds of the invention can be applied to the plant or soil using anyof a variety of known protocols. The present compounds can be used aloneor in combination with other sprays, fertilizers or injections.

Specific in vitro uses of the present compounds include facilitatingadaptation to culture, maintenance of differentiation and function,stimulating cellular productivity and increasing cellular vitality. Celltypes susceptable to the effects of the present compounds includeeucaryotic cells, for example, from mammalian tissue or from insect orarachnid tissue, as well as cells derived from plant tissue and fungal(eg yeast) cells. Procaryotic cells, including bacterial cells, can alsobe used.

Cells can be grown or stored in the presence of the present compoundsusing any of a variety of available technique, including growth onplastic or glass or other support (eg beads or hollow fibers), growth insuspension (eg in liquid or semisolid medium), growth in a bioreactor,or storage in a frozen or dried state. Primary cultures or serialcultures, or otherwise, can be used.

As indicated above, the compounds of the invention can be used, asappropriate, to alter cell viability/vitality and/or productivity.Altered viability/vitality can be manifest, for example, as a delay insenescence or altered adaptability to culture. Altered productivity canbe manifest, for example, by an alteration in the production of a cellspecific product, eg a protein, for example, an alteration in hybridomaproduction of antibodies. The compounds of the invention can also beused to stimulate host cell production of recombinant or naturalproteins or activities. The compounds of the invention can also be usedin whole cell or cell free environments to stimulate or controlbiochemical reactions including replication, transcription, translation,transport or modification of structural or enzymatically activemolecules or organelles.

The amount of the compound of the invention to be used and the frequencyof exposure can be readily determined by one skilled in the art and willvary with the cell type, the compound used and the effect sought. Indetermining optimum concentrations, appropriate in vitro assays are runin the femtogram/ml to 10's of mg/ml range.

Various aspects of the present invention are -described in greaterdetail in the non-limiting Examples that follow. Certain of thesynthetic procedures described below correspond to those described byKnight et al, Cancer Research 54:5623 (1994) or in U.S. Pat. No.4,218,404, or represent modifications thereof. In addition, thedisclosures of WO 92/00955 and PCT/US91/04725 are relevant here,including the portions therein that relate to syntheses, therapeuticregimens and cell culture treatment protocols, those regimens andprotocols being applicable to the compounds of the present invention.

EXAMPLE I Synthesis of N-Carbobenzoxy-β-Alanyl-Taurine Zinc Salt

Method I

Preparation of N,N'-bis (CBZ)-β-Alethine from N-CBZ-β-alanine

To a 250 ml round bottom flask were added a stir bar, N-CBZ-alanine(5.805 g, 26.008 mmol), N-hydroxysuccinimide (2.993 g, 26.008 mmol, 1eq.), and 1,3-dicyclohexylcarbodiimide (5.366 g, 26.008 mmol, 1 eq.).The flask was sealed with a septum and purged with argon. CH₂ Cl₂ (86ml) was then added and the mixture stirred overnight at room temperature(rt). All of the solids did not dissolve upon addition of CH₂ Cl₂. Thesolids were then removed via vacuum filtration through apre-argon-purged medium glass fritted buchner funnel. The funnel wasequipped with an argon purge funnel and a 500 ml round bottom flaskcontaining a stir bar and cystamine.2HCl (1.464 g, 6.502 mmol, 0.25eq.). The flask and solids (white) were then rinsed with 3×15 ml CH₂Cl₂. The filtrate was colorless to light yellow. The flask was removedfrom the buchner, sealed with a septum and purged with argon. To thestirring solution was added Et₃ N (2.9 ml, 20.806 mmol, 0.8 eq.). All ofthe solids did not dissolve. The reaction was stirred overnight at roomtemperature. The product was then collected using an 11 cm buchnerfunnel with #541 Whatman filter paper. The flask and solids (white) wererinsed with 3×15 ml CH₂ Cl₂. The filtrate was colorless to yellow. Thesolids were placed in a 250 ml round bottom flask and dried under highvacuum overnight. The crude product weight was determined and DMSO (0.3g/ml) was added and heated to 70° C.-90° C. to dissolve the solids withthe aid of stirring. H₂ O (0.12 g/ml) was then added slowly withvigorous stirring. The mixture was cooled to room temperature andcollected after 3 hours using an 11 cm buchner funnel with #541 Whatmanfilter paper. The solids (white) and flask were rinsed 3×15 ml CH₂ Cl₂H₂ O followed by 2×15 ml EtOAc. The solids were chopped-up with aspatula and dried under high vacuum in a 250 ml round bottom flask. Therecovery was 3.568 g corresponding to a 97.5% yield.

Preparation of N-carbobenzoxy-β-alanyl-taurine zinc salt fromN,N'-bis(CBZ)-β-alethine (small additions of ZnO or Ca(OH)₂)

To a 250 ml erlenmeyer flask were added a stir bar,N,N'-bis(CBZ)β-alethine (2.524 g, 4.486 mmol), dimethylsulfoxide (2.5ml), N,N-dimethylformamide (2.5 ml), pyridine (3.2 ml), CHCl₃ (75 ml),and H₂ O (150 ml). The mixture was stirred vigorously giving an emulsion(not all solids dissolved). A pH meter was immersed in the aqueousphase. The pH was near 7.3 to 7.7. I₂ (7.97 g, 31.401 mmol, 7 eq.) wasthen added. Initially the organic phase was red and the aqueous phasewas colorless. During the reaction, the color of the aqueous phasedarkened to red and the emulsion subsided. The pH dropped to 5.7 within10 minutes of adding I₂. ZnO (100-200 mg, 0.3-0.6 eq.) was added inportions to keep the pH between 5.7 and 6.0. After ˜3.5 hours, the pHstabilized and the reaction was allowed to stir for an additional 2hours (5.5 h total reaction time). The phases were separated (organicwas dark red) and the aqueous phase was washed with 10 ml CHCl₃. Theaqueous phase (light red) was extracted additionally with CHCl₃ using acontinuous liquid/liquid extractor overnight. The aqueous phase(colorless to very light pink) was separated, partially evaporated on arotary evaporator to remove dissolved organics, shelf frozen, andlyophilized. The residue (golden brown) was dissolved in 1 ml H₂ O and 3ml acetonitrile and added to 100 ml acetonitrile. The white precipitatewas collected on a #541 Whatman filter paper and rinsed with 40 mlacetonitrile. The recovery was 1.676 g of white solids corresponding toa 52% yield (N-carbobenzoxy-β-alanyl-taurine zinc salt).

Preparation off N-carbobenzoxy-β-alanyl-taurine zinc salt fromN,N'-bis(CBZ)β-alethine (ZnO added initially)

To a 125 ml erlenmeyer flask were added a stir bar,N,N'-bis(CBZ)β-alethine (809 mg, 1.438 mmol), dimethylsulfoxide (0.8ml), N,N-dimethylformamide (0.8 ml), pyridine (1.0 ml), CHCl₃ (24 ml),H₂ O (80 ml), and ZnO (526 mg, 6.470 mmol, 4.5 eq.). The mixture wasstirred vigorously giving an emulsion (not all solids dissolved). I₂(3.28 g, 12.904 mmol, 9 eq.) was then added. Initially, the organicphase was red and the aqueous phase was colorless. During this reaction,the color of the aqueous phase darkened to red and the emulsionsubsided. The mixture was stirred overnight. The phases were thenseparated (organic was dark red) and the aqueous washed with 20 mlCHCl₃. The aqueous phase (light red) was extracted additionally withCHCl₃ using a continuous liquid/liquid extractor overnight. The aqueousphase (colorless to very light pink) was separated, partially evaporatedon a rotary evaporator to remove dissolved organics, shelf frozen, andlyophilized. The residue (golden brown) was dissolved in 0.5 ml H₂ O and2 ml acetonitrile and added to 75 ml acetonitrile. The white precipitatewas collected on a #541 Whatman filter paper and rinsed with 20 mlacetonitrile. The recovery was 630 mg of white solids corresponding to a61% yield (N-carbobenzoxy-β-alanyl-taurine zinc salt).

The ¹³ C NMR spectral data were as follows (for numerical assignment seeFIG. 2A):

    ______________________________________                                        Signal    DMSO solvent: C--H coupled                                          ______________________________________                                        1         48.4                                                                  2 33.8                                                                        3 172.2                                                                       4 34.6                                                                        5 36.4                                                                        6 156.4                                                                       7 65.4                                                                        8 135.0                                                                       9 125.8                                                                       10 126.5                                                                      11 128.0                                                                    ______________________________________                                    

Method IIa

Preparation of N-carbobenzoxy-β-alanyl-taurine (free acid and zinc salt)from N-(CBZ)-β-alanine

The N-(CBZ)-β-alanine (563 mg, 2.522 mmol), N-hydroxysuccinimide (290mg, 2.522 mmol) and DCC (520 mg, 2.522 mmol) were dissolved (no obviousdissolution) in CH₂ Cl₂ (11.5 ml, to make a 0.22 M solution). Thereaction was allowed to mix overnight at room temperature. The crudereaction mixture was filtered through a sintered glass funnel to removethe dicyclohexylurea (DCU). The reaction was filtered "anhydrously" intoa flask containing 316 mg (2.522 mmol) taurine. The filter cake waswashed with 3, 5 ml volumes of CH₂ Cl₂. After adding 316 μl Et₃ N (1eq), the reaction was allowed to mix at room temperature. The reactionwas allowed to mix until complete by NMR. The crude reaction mixture waspurified by trituration with MeCN. The crude reaction mixture wasdissolved in 14 ml CH₂ Cl₂ (0.2 M) and 1 eq of triflic acid was added.The reaction was allowed to mix overnight at room temperature althoughthe reaction appeared complete after mixing for only 15-20 minutes. Thereaction mixture was filtered and the filter cake washed with CH₂ Cl₂.The filter cake (the free acid) was divided into two portions. Oneportion (305 mg) and 0.5 eq Zn(OH)₂ were dissolved in 5 ml H₂ O andallowed to mix for 1 hour and then it was concentrated by lyophilizationto give 340 mg as a white solid (66% based on startingN-(CBZ)-β-alanine). The other portion was purified and characterized asthe free acid (190 mg). NMR data were obtained on both the free acid andthe Zn salt, the Zn salt being the more pure (see FIG. 1 for reactionscheme).

The ¹ H NMR spectral data were as follows (for alphabetical assignmentsee FIG. 2B):

    ______________________________________                                        Signal        D.sub.2 O solvent                                               ______________________________________                                        a             2.99 (t, J = 12.8 Hz, 2H)                                         b 3.48 (t, J = 12.8 Hz, 2H)                                                   c not seen due to hydrogen bonding                                            d 2.38 (t, J = 12.4 HZ, 2H)                                                   e 3.35 (m, 2H)                                                                f not seen due to hydrogen bonding                                            g 5.07 (m, 4H)                                                              ______________________________________                                    

The ¹³ C NMR spectral data were as follows (for numerical assignment seeFIG. 2A):

    ______________________________________                                               Signal                                                                              DMSO solvent                                                     ______________________________________                                               1     51.1                                                               2 36.1                                                                        3 170.2                                                                       4 36.4                                                                        5 37.7                                                                        6 156.6                                                                       7 65.7                                                                        8 137.8                                                                       9 128.3                                                                       10 128.9                                                                      11 128.9                                                                    ______________________________________                                    

Method IIb

Preparation of N-carbobenzoxy-β-alanyl-taurine (Free Acid and Zinc Salt)from N-(CBZ)-β-Alanine (Scale up)

In a three-neck 1 L flask was placed CBZ-β-alethine (48.2 g, 215.9 mmol)under N₂. To this was added freshly distilled methylene chloride (750mL), followed by N-hydroxysuccinimide (24.85 g, 215.9 mmol). To theresulting suspension was added 1,3-dicyclohexylcarbodiimide (DCC, 44.54g, 215.9 mmol). At this scale the reaction generated a noticeableexotherm, sufficient to reflux the CH₂ Cl₂. The reaction mixture wasstirred under N₂ for 5 hours at which point the mixture was filteredthrough a sintered glass buchner funnel. The filter cake was washed withCH₂ Cl₂ (3×100 mL). To the filtrate was added taurine (27.03 g, 215.9mmol) and triethylamine (33.1 mL, 237.5 mmol). The reaction was stirredunder N₂ and monitored by ¹ H-NMR analysis. The reaction mixture wasvacuum filtered through a buchner funnel using Whatman #542 filterpaper. The filtrate was stripped to an oil using reduced pressure, thenplaced on high vacuum. The "oil" was triturated with acetonitrile withone drop of water to quench any unreacted DCC. The mixture was filteredand the MeCN was stripped off under reduced pressure then placed on highvacuum. The resulting oil was dissolved in water (50 mL). A curdy whiteprecipitate formed, more water (150 mL) was added and the resultingsolid was filtered off. In the filtrate an oil precipitated out ofsolution. ¹ H-NMR spectra were obtained to determine the location ofproduct. The product was in the aqueous layer as expected. The aqueousphase containing the product was then eluted through a H+ ion exchangecolumn. Fractions (225 mL) were collected and spotted on TLC. Thedesired product was found in fractions 2-7. These fractions werecombined and the water removed under reduced pressure. To the resultingoil was added MeCN (1 L) and the solution was stirred. The remainingwater was removed by azeotropic distillation with the MeCN. Theresulting solid was collected by vacuum filtration and washed with MeCN.The solid was vacuum dried in a 1 L round bottom flask then transferredto a tared 4 oz. amber bottle. Final package weight was 38.89 g (117.7mmol, 54.5% yield). The zinc salt was prepared by treatment with Zn(OH)₂in H₂ O, followed by lyophilization. The spectral data for the productmatched exactly a standard sample of N-carbobenzoxy-β-alanyl-taurinezinc salt.

EXAMPLE II Synthesis of β-Alanyl-Taurine (Free Acid and Zinc Salt)

N-carbobenzoxy-β-alanyl-taurine (1.00 g, 3.4 mmol) was slurried in 23 mlglacial AcOH. To the mixture was added 3.4 mL HBr in AcOH (30 wt %) toresult in a clear solution. The reaction was heated to 40° C. andallowed to mix overnight. The product precipitated out of solution andacetonitrile was added to force the precipitation. The mixture wasfiltered, the filter cake washed and the product collected. The crude Brsalt was loaded onto an ion exchange column (Dowex AG1-XB8). The columnwas eluted with H₂ O. The product cut was collected and lyophilized togive 583 mg of β-alanyl-taurine (87.3%). The zinc salt was prepared bytreatment with Zn(OH)₂ in H₂ O, followed by lyophilization. (See FIG. 1for reaction Scheme.)

The ¹ H NMR spectral data were as follows (for alphabetical assignmentsee FIG. 2C):

    ______________________________________                                        Signal        D.sub.2 O solvent                                               ______________________________________                                        a             3.09 (t, J = 12 Hz, 2H)                                           b 3.59 (t, J = 12 Hz, 2H)                                                     c not seen due to hydrogen bonding                                            d 2.66 (t, J = 12 Hz, 2H)                                                     e 3.25 (t, J = 12 Hz, 2H)                                                   ______________________________________                                    

The ¹³ C NMR spectral data were as follows (for numerical assignment seeFIG. 2D):

    ______________________________________                                               Signal                                                                              DMSO solvent                                                     ______________________________________                                               1     50.8                                                               2 36.2                                                                        3 169.6                                                                       4 33.1                                                                        5 36.2                                                                      ______________________________________                                    

For comparison, β-alanyl-taurine zinc salt prepared using the method ofKnight et al, Cancer Research 54:5623 (1994) gave the following ¹ H NMRspectra (for alphabetical assignment see FIG. 2C):

    ______________________________________                                        Signal        D.sub.2 O solvent                                               ______________________________________                                        a             2.93 (t, J = 12 Hz, 2H)                                           b 3.42 (t, J = 12 Hz, 2H)                                                     c not seen due to hydrogen bonding                                            d 2.50 (t, J = 12 Hz, 2H)                                                     e 3.10 (t, J = 12 Hz, 2H)                                                   ______________________________________                                    

EXAMPLE III

Preparation of N-Carbobenzoxy-β-Alanyl-Ethanolamine Phosphate (Free Acidand Zinc Salt) from N-(CBZ)-β-Alanine

N-(CBZ)-β-alanine (274 mg, 1.23 mmol), N-hydroxysuccinimide (141 mg,1.23 mmol) and dicyclohexylurea (DCC, 253 mg, 1.23 mmol) were dissolvedin tetrahydrofuran (THF, 4.1 mL). The reaction was allowed to mix atroom temperatore overnight before being filtered to remove thedicyclohexylurea (DCU). To the filtrate, a solution of 2-aminoethyldihydrogen phosphate (1.23 mmol) in H₂ O (0.5 ml) was added. To thereaction mixture was added 2.1 molar equivalents of triethylamine. Thereaction was allowed to mix for three days before the THR was removedunder vacuum. The remaining aqueous phase was filtered and loaded onto aprepared ion exchange column (Dowex AG 50W-X8). The column was elutedwith water. The product fractions were collected and lyophilized. Thecrude solid (260 mg) was treated with 1.0 molar equivalents of Zn(OH)₂in H₂ O to make the salt. The crude solid (after lyophilization) wastriturated with acetronile and collected (50 mg).

EXAMPLE IV Synthesis of N-Carbobenzoxy-β-Alanyl-AminoethylphosphonicAcid (Free Acid and Zinc Salt) from N-(CBZ)-β-Alanine

N-(CBZ)-β-alanine (301 mg, 1.35 mmol), N-hydroxysuccinimide (155 mg,1.35 mmol) and dicyclohexylurea (DCC, 278 mg, 1.35 mmol) were dissolvedin tetrahydrofuran (THF, 4.5 ml). The reaction was allowed to mix atroom temperature overnight before being filtered to remove thedicyclohexylurea (DCU). To the filtrate, a solution of2-aminoethylphosphonic acid (1.35 mmol) in H₂ O (0.5 ml) was added. Tothe reaction mixture was added 2.1 molar equivalents of triethylamine.The reaction was allowed to mix for three days before the THF wasremoved under vacuum. The remaining aqueous phase was filtered andloaded onto a prepared ion exchange column (Dowex AG 50W-X8). The columnwas eluted with water. The product fractions were collected andlyophilized. The crude solid (270 mg) was treated with 1.0 molarequivalents of Zn(OH)₂ in H₂ O to make the salt. The crude solid (afterlyophilization) was triturated with acetonitrile and collected (50 mg).

EXAMPLE V In Vitro Simulation of Differentiation and Production ofDifferentiated Product by N-Carbobenzoxy-β-Alanyl-Taurine Zinc Salt

Hybridoma cells (ATCC #CRL-8014, OKT-8, secreting an IgG2 anti-humanT-cell subset antibody) were grown with or withoutN-carbobenzoxy-β-alanyl-taurine zinc salt in T25 culture flasks. Cellswere inoculated at a density of 10,000 cells/ml and maintained below500,000 cells/ml in 5 mls of protein-free media containing HyQ-PF-MABfrom Hyclone. Aliquots were assayed for Mab production by a sandwichELISA. Aliquots were diluted to be within the standard range and addedto plates precoated with goat anti-mouse IgG by incubating two hours atroom temperature. Wells were washed and reacted with dilutedsupernatants, then washed and detected with peroxidase labeledanti-mouse antibodies. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Altered Production of Monoclonal Antibodies                                     from Hybridomas                                                                  Drug           1 gG, μg/ml                                                                          IgG, pg/cell                                    ______________________________________                                        0 - control     12        41.7                                                  N-carbobenzoxy- 50 80.6                                                       β-alanyl-taurine                                                         200 pg/ml                                                                     Increase 316% 93%                                                           ______________________________________                                    

EXAMPLE VI Stimulation of Protein Production from Mammalian Cells

CHO cells containing a cloned gene for tissue plasminogen activator(tPA) were obtained from ATCC as #CRL-9606. The tPA gene had beenintroduced by transfection of a plasmid pETPFR. The cells werepropagated in T-25 flasks in Ham's F-12 medium with 10% fetal bovineserum. The cell cultures were innoculated by adding 5 ml of cells at2×10⁴ cells/ml into T-25 flasks.

The effect of N-carbobenzoxy-β-alanyl taurine zinc salt on theproduction of tPA was tested by adding this compound to the growthmedium, and maintaining the indicated levels of the compound over many(eg 6) passages of the cells.

At the end of log phase growth, the cultures were harvested bytrypsinization. One ml was centrifuged to remove cells and thesupernatant assayed for tPA in the IMUBIND total tPA Stripwell ELISAfrom American Diagnostica Inc.

The results shown in Table 2 demonstrate that the compound(N-carbobenzoxy-β-alanyl taurine zinc salt, #'s 1, 2 and 3 in Table 2)alters the per-cell production of tPA by about 2 fold. The cell numbersper milliliter were determined by direct counting of trypan-blue stainedcells on a hemacytometer.

                  TABLE 2                                                         ______________________________________                                                         Supernatant                                                    Com- ELISA Optical Concentration Culture Cell Picograms                       pound Density nanograms/ml* Density tPA per cell                            ______________________________________                                        Control                                                                             0.177        424.6    2.2 × 10.sup.5                                                                  1.93                                        #1 0.332 1,492.8 4.0 × 10.sup.5 3.73                                    1 pg/ml  [3.5 × control]  [1.9 × control]                         #2 0.317 1,389.4 4.8 × 10.sup.5 2.89                                    1 pg/ml  [3.3 × control]  [1.5 × control]                         #3 0.422 2,113.0 5.2 × 10.sup.5 4.06                                    1 pg/ml  [5.0 × control]  [2.1 × control]                       ______________________________________                                         *The standard curve is "Abs = 0.1154 + .029* Concentration", with a           correlation coefficient of 0.9977.                                       

EXAMPLE VII In Vivo Coordinated T Cell Dependent Response Delayed TypeHypersensitivity

In order to test an in vivo coordinated T cell dependent response,delayed type hypersensitivity (DTH) was measured. DTH is the test usedclincially to determine if a person has mounted an immune response tomany antigens. The most frequent application is measurement of DTH inresponse to tuberculosis called a PPD or tine test. It is also used todetermine if a patient, such as a cancer patient, has had a failure ofthe immune system and become anergic.

In the laboratory this test involves the generation of a specificresponse to a oxazalone (OX) in mice and measurement of the response.The generation of the response (sensitization or initial exposure) wascaused by the application of OX to the shaved abdomen of the animal (50μl of 1.2% OX in olive oil). Measurement of the response occurred 24 and48 hours following application of OX (5 μl 1.2%) to the right ear of theanimal. In the experiment performed, sensitization occurred on day 0;challenge occurred on day 4. On days five and six, the thickness of theear was measured and the thickness before challenge was subtracted. Thedoses of OX used were chosen so that some normal untreated animals had aperceptible but moderate response to the OX at 24 hrs.

In order to measure the effect of N-carbobenzoxy-β-alanyl-taurine zincsalt, various doses were injected iv on Day -2, 0, +2 and +4.

Ten animals were used per group. Drug dilutions are made up and coded atone facility and an independent contract facility (Midlantic Research)performed all procedures in a "blind" fashion. In no case did controlears (those either not sensitized or not challenged) have swelling over40 μ. Animals with 50 μ to 99 μ swelling were rated as having moderateswelling. Three control (saline injected) animals mounted a moderateresponse on at 24hrs as expected. N-carbobenzoxy-β-alanyl taurinealtered the response rate to eight of ten in two different treatmentgroups (this is above the 95% confidence bounds for an altered responseby relative risk measure). All groups of animals receiving between 1fg/kg and 1 mg/kg had more moderate responders than the saline injectedgroup.

Those animals with 100 μ or greater swelling were rated as having majorimmune responses. No saline injected animal had major immune response at24 hours while a total of 14 N-carbobenzoxy-β-alanyl-taurine treatedanimals had major immune response. One non drug treated animal had amajor response at either 24 or 48 hours post challenge, while 21N-carbobenzoxy-β-alanyl-taurine treated animals had a major response atone of these times. The data are presented in Table 3.

                  TABLE 3                                                         ______________________________________                                        N-carbobenzoxy-β-alanyl taurine increases DTH                              Percent of Animals with DTH                                                   Dose/kg >   0     1 fg 32 fg                                                                              1 pg 32 pg                                                                              1 ng 1 μg                                                                            1 mg                        ______________________________________                                        Moderate DTH at                                                                         30    70     60   50    80*  80* 40   70                              24 hrs                                                                        Major DTH at 0 20 20 10 10 20 10  50*                                         24 hrs                                                                        Major DTH at 48 0 20 20 10 10 20 10 50                                        hrs.                                                                          Major OTH at 10 20 20 10 40 20 30 60                                          either 24 or 48                                                               hrs.                                                                        ______________________________________                                         *Relative Risk (RR) compared to control exceeds 1 even at lowest end of       95% confidence bounds. RR not tested at 48 hours                         

EXAMPLE VIII Blood Cell Stimulation

A 96-well-based suspension culture system (Warren et al, Stem Cells13:167 (1995) for human hematopoietic progenitor cells was used tomonitor the commitment and differentiation of CD34+ cells in vitro.Expression of maturation and lineage markers on the cells in culturewere measured by ELISA. The CD34+ cells were isolated from umbilicalcord blood (90% purity) and grown in liquid culture in 96-well plates(2000 per well) for 10 days. A combination of growth factors was addedthat stimulates the expression of the appropriate lineage markers. Theculture consisted of: IMDM plus 15% FBS, 0.5 ng/ml IL-3, 20 ng/ml SCF, 1unit/ml EPO, 1 ng/ml GCSF and the indicated concentrations of testcompounds. The cells were then fixed with aglutaraldehyde-paraformaldehyde mixture, attaching the cells firmly tothe plastic. An ELISA was performed (Warren et al, Stem Cells 13:167(1995)), using appropriate primary antibodies directed against cellsurface markers. The expression of three different lineage markers wasmeasured: CD14 (monocyte), CD15 (neutrophil), and glycophorin A(erythroid). The results are presented in Table 4.

                  TABLE 4                                                         ______________________________________                                        Increase in Blood Cell Production                                               Percent difference (relative to control)                                      Study 1: 1 ng/ml compound                                                                           red                                                     Com- mono- neutro- blood Study 2: 1 μg/ml                                pound   cytes   phils   cells                                                                              CD34 cells                                                                            monocytes                                                                            RBC                               ______________________________________                                        Taurox- 18      14      1     0      2      4                                   BP                                                                            Taurox- 18 21 0 33 9 55                                                       BOP                                                                           Taurox- 15 0 0 not tested                                                     SB                                                                            Taurox-S 47 55 50 not tested                                                ______________________________________                                         Taurox-BP = Ncarboxybenzoxy-alanyl aminoethylphosphonic acid                  TauroxBOP = Ncarboxybenzoxy-alanyl-ethanolamine phosphate                     TauroxSB = Ncarboxybenzoxy-alanyl-taurine                                     TauroxS = alanyl-taurine                                                      Study 2: same, expect 1700 cells per well                                

EXAMPLE IX Immune Stimulation

BALB/c female mice (4-5 weeks of age) were prebled, then injected ipwith 0.1 mls of the indicated compounds on day -7, day -5, day -3, andday 0. On day 0, they received soluble polysaccharide antigen Pn14polysaccharide conjugated to Tetanus Toxoid, 10 μg in 0.1 mls, given ip.Two other groups received Pn14-TT, 10 μg in 0.1 mls of a 60% emulsion ofcomplete Freund's adjuvant (CFA) on Day 0, given subcutaneously abovethe hind leg, just off the midline. The two CFA groups were treatedidentically. All mice were bled on Day 4 and Day 14. On Day 84, micewere bled and boosted with 5 μg Pn14 (not conjugated to TT) and eitherthe experimental compound or incomplete Freund's adjuvant (for thosepreviously given CFA). Mice were bled on day 94. Sera at 1:1000 wereanalyzed by ELISA for anti-Pn14 antibodies. Prebleed values weresubtracted.

    ______________________________________                                        Mean Change in O.D. (Optical density, indicating presence of antibody)                       Change                                                                                       from pretest to 4 & Change from pre-boost                                     14 days after in- 10 days after boost with                                    jection with antigen UNCONJUGATED               Compound  Day 4     Day 14  polysaccharide                                    ______________________________________                                        CFA (control)                                                                           -0.6      672     -256                                                CFA (control) -1.0 367 not tested                                             Taurox-BP, ng 15.5 1399 not tested                                            Taurox-BP, ug 6.5 616 not tested                                              Taurox-BP, mg 3.3 924 187                                                     Taurox-BOP, ng 5.4 877 not tested                                             Taurox-BOP, ug 1.8 515 not tested                                             Taurox-BOP, mg 0 366 not tested                                               Taurox-SB, ng 6.2 705 not tested                                              Taurox-SB, ug 3.9 483 not tested                                              Taurox-SB, mg 1.1 697 139                                                     Taurox-S, ng 13.1 681 204                                                     Taurox-S, ug 3.6 671 not tested                                               Taurox-S, mg 2.9 697 not tested                                             ______________________________________                                         Notes                                                                         1. Values are the mean of 3 animals per drug/dose group.                      2. Doses were 5 ng per kilogram of animal, 5 ug/kg, and 5 mg/kg of test       compounds given prior to and with conjugated antigen, indicated above as      "ng", "ug", and "mg".                                                         3. CFA  Complete Freund's adjuvant, the current "gold standard" vaccine       adjuvant, but approved only for animal uses due to its toxicity. Two          groups of 3 animals were used. Published data indicate CFA stimulates         response 10× compared to saline.                                        Discussion                                                                    a. Only in animals treated with a compound of this invention is a 4day        response seen.                                                                b. Only in animals treated with a compound of this invention is a respons     to unconjugated polysaccharide seen.                                          c. The 14day response is greater with treatment.                         

EXAMPLE X Syntheses

Synthesis of N-Carbobenzoxy-β-Alanyl-Ethanolamine Sulfate--Taurox BOS:

To a THF solution of N-(CBZ)-β-alanine and N-hydroxysuccinimide, addDCC. The reaction is mixed overnight at room temperature. The crudereaction mixture is filtered through a sintered glass funnel into around bottom flask to remove the DCU that is formed. The activated estershould remain in solution. After concentrating and redissolving insolvent, ethanolamine or an alcohol protected derivative can be added asa solution to the solution containing the activated ester. Triethylaminecan also be added. Workup of the reaction and purification results information of N-carbobenzoxy-β-alanyl-ethanolamine. The free alcohol canbe sulfated by a variety of methods to result in formation ofN-carbobenzoxy-β-alanyl-ethanolamine sulfate.

Synthesis of β-Alanyl-Ethanolamine Sulfate (fromN-Carbobenzoxy-β-Alanyl-Ethanolamine Sulfate)--Taurox OS:

In a similar fashion to the conversion ofN-carbobenzoxy-β-alanyl-taurine to β-alanyl-taurine,N-carbobenzoxy-β-alanyl-ethanolamine sulfate can be converted toβ-alanyl-ethanolamine sulfate. The conversion can be effected byslurrying the N-carbobenzoxy-β-alanyl-ethanolamine sulfate in glacialAcOH. To the mixture, HBr in AcOH (30 wt %) is added. The reaction canbe heated and allowed to mix for a period of not less than 1 hour. Theproduct can be isolated by usual workup and precipitation.

Synthesis of β-Alanyl-Ethanolamine Phosphate (fromN-Carbobenzoxy-β-Alanyl-Ethanolamine Phosphate)--Taurox OP

In a similar fashion to the conversion ofN-carbobenzoxy-β-alanyl-taurine to β-alanyl-taurine,N-carbobenzoxy-β-alanyl-ethanolamine phosphate can be converted toβ-alanyl-ethanolamine phosphate. The conversion can be effected byslurrying the N-carbobenzoxy-β-alanyl-ethanolamine phosphate in glacialAcOH. To the mixture, HBr in AcOH (30 wt %) is added. The reaction canbe heated and allowed to mix for a period of not less than 1 hour. Theproduct can be isolated by usual workup and precipitation.

Synthesis of β-Alanyl-Aminoethylphosphonic Acid (fromN-Carbobenzoxy-β-Alanyl-Aminoethylphosphonic Acid)--Taurox P

In a similar fashion to the conversion ofN-carbobenzoxy-β-alanyl-taurine to β-alanyl-taurine,N-carbobenzoxy-β-alanyl-aminoethylphosphonic acid can be converted toβ-alanyl-aminoethylphosphonic acid. The conversion can be effected byslurrying the N-carbobenzoxy-β-alanyl-aminoethylphosphonic acid inglacial AcOH. To the mixture, HBr in AcOH (30 wt %) is added. Thereaction can be heated and allowed to mix for a period of not less than1 hour. The product can be isolated by usual workup and precipitation.

Structures of the above compounds are shown in FIG. 3.

All documents cited above are hereby incorporated in their entirety byreference. The entire contents of U.S. Provisional Appln. 60/005,336,filed Oct. 17, 1995, is incorporated herein by reference.

One skilled in the art will appreciate from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention.

What is claimed is:
 1. A method of enhancing the IgG immune response toone or more antigens comprising administering to a mammal in need ofsuch enhancement an amount of said one or more antigens and a compoundof formula (I): ##STR3## wherein: A is a group of the formula --PO₃ H,--SO₃ H, --OPO--(OH)₂ --OSO₂ OH, or --SH, or pharmaceutically acceptablesalt thereof or physiologically hydrolyzable derivative thereof, ordisulfide thereof when A is --SH,R₁ is H, a linear or branched lowerallyl, an arylalkyl or an alkenyl, R₂ is H, a linear or branched loweralkyl, an alkenyl, an arylalkyl, an acyl, a carbonate ester, an allyloxycarbonyl, a cycloalkoxycarbonyl, an unsubstituted arylalkoxycarbonyl ora substituted arylalkoxycarbonyl, or R¹ and R² taken together form, withthe nitrogen to which they are attached, a 5 to 7 membered ring, and L¹and L² are, independently, a hydrocarbon linking group, a cycloalkyl, oran interphenylene, sufficient to enhance the IgG immune response to saidone or more antigens.
 2. The method of claim 1 whereinA is a group ofthe formula --PO₃ H, --SO₃ H, --OPO--(OH)₂, --OSO₂ OH, or --SH, orpharmaceutically acceptable salt thereof or physiologically hydrolyzable(C₁ -C₄)alkyl or arylalkyl ester thereof, or disulfide thereof when A is--SH; R₁ is H, a linear or branched (C₁ to C₆) alkyl, a substituted orunsubstituted phenyl (C₁ -₄) alkyl, or a (C₂ -C₆) alkenyl; R₂ is H, alinear or branched (C₁ to C₆) alkyl, a (C₂ -C₆) alkenyl, a substitutedor unsubstituted phenyl (C₁ -C₄) alkyl, an acetyl, benzoyl orarylsulfonyl, a (C₁ -C₇) alkoxycarbonyl, --OCOCH₂ CH═CH₂, a (C₃ -C₈)cyclo(C₁ -C₈) alkoxycarbonyl, --OCOCH₂ C₆ H₅ or a substitutedarylalkoxycarbonyl wherein the substituent is a halogen, a nitro group,an amino group or a methoxyl group; or R¹ and R² taken together form,with the nitrogen to which they are attached, a 5 to 7 membered ring;and L¹ and L² are, independently, a linear or branched chain alkyl ofthe formula --(C_(n) H_(2n))-- wherein n is 1 to 8; a cycloalkyl of 3 to8 carbon atoms, or an interphenylene.
 3. A vaccine comprising one ormore antigens and a compound of the formula ##STR4## wherein: A is agroup of the formula --PO₃ H, --SO₃ H, --OPO--(OH)₂ --OSO₂ OH, or --SH,or pharmaceutically acceptable salt thereof or physiologicallyhydrolyzable derivative thereof, or disulfide thereof when A is --SH,R₁is H, a linear or branched lower alkyl, an arylalkyl or an alkenyl, R₂is H, a linear or branched lower alkyl, an alkenyl, an arylalkyl, anacyl, a carbonate ester, an alkyloxy carbonyl, a cycloalkoxycarbonyl, anunsubstituted arylalkoxycarbonyl or a substituted arylalkoxycarbonyl, orR¹ and R² taken together form, with the nitrogen to which they areattached, a 5 to 7 membered ring, and L¹ and L² are, independently, ahydrocarbon linking group, a cycloalkyl, or an interphenylene, whereinthe compound enchances the production of antigen-specific IgG in amammal immunized with said vaccine.
 4. The vaccine of claim 3, whereinAis a group of the formula --PO₃ H, --SO₃ H, --OPO--(OH)₂, --OSO₂ OH, or--SH, or pharmaceutically acceptable salt thereof or physiologicallyhydrolyzable (C₁ -C₄)aklyl or arylalkyl ester thereof, or disulfifdethereof when A is --SH; R₁ is H, a linear or branched (C₁ to C₆)alkyl, asubstituted or unsubstituted phenyl(C₁ -C₄)alkyl, or a (C₂ -C₆)alkenyl;R₂ is H, a linear or branched (C₁ to C₆)alkyl, a (C₂ -C₆)alkenyl, asubstituted or unsubstituted phenyl (C₁ -C₄)alkyl, an acetyl, benzoyl orarylsulfonyl, a (C₁ -C₇)alkoxycarbonyl, --OCOCH₂ CH═CH₂, a (C₃-C₈)cyclo(C₁ -C₈)alkoxycarbonyl, --OCOCH₂ C₆ H₅ or a substitutedarylalkoxycarbonyl wherein the substituent is a halogen, a nitro group,an amino group or a methoxyl group; or R¹ and R² taken together form,with the nitrogen to which they are attached, a 5 to 7 membered ring;and L¹ and L² are, independently, a linear or branched chain alkyl ofthe formula --(C_(n) H_(2n))-- wherein n is 1 to 8; a cycloalkyl of 3 to8 carbon atoms, or an interphenylene.
 5. A method of enhancing theproduction of antigen-specific IgG in a mammal comprising administeringto the mammal an amount of one or more antigens and a compound offormula I: ##STR5## wherein: A is a group of the formula --PO₃ H, --SO₃H, --OPO--(OH)₂ --OSO₂ OH, or --SH, or pharmaceutically acceptable saltthereof or physiologically hydrolyzable derivative thereof, or disulfidethereof when A is --SH,R₁ is H, a linear or branched lower alkyl, anarylalkyl or an alkenyl, R₂ is H, a linear or branched lower alkyl, analkenyl, an arylalkyl, an acyl, a carbonate ester, an alkyloxy carbonyl,a cycloalkoxycarbonyl, an unsubstituted arylalkoxycarbonyl or asubstituted arylalkoxycarbonyl, or R¹ and R² taken together form, withthe nitrogen to which they are attached, a 5 to 7 membered ring, and L¹and L² are, independently, a hydrocarbon linking group, a cycloalkyl, oran interphenylene, sufficient to enhance said production ofantigen-specific IgG.
 6. The method of claim 5 whereinA is a group ofthe formula --PO₃ H, --SO₃ H, --OPO--(OH)₂, --OSO₂ OH, or --SH, orpharmaceutically acceptable salt thereof or physiologically hydrolyzable(C₁ -C₄)alkyl or arylalkyl ester thereof, or disulfide thereof when A is--SH; R₁ is H, a linear or branched (C₁ to C₆) alkyl, a substituted orunsubstituted phenyl(C₁ -C₄) alkyl, or a (C₂ -C₆) alkenyl; R₂ is H, alinear or branched (C₁ to C₆) alkyl, a (C₂ -C₆) alkenyl, a substitutedor unsubstituted phenyl (C₁ -C₄) alkyl, an acetyl, benzoyl orarylsulfonyl, a (C₁ -C₇) alkoxycarbonyl, --OCOCH₂ CH═CH₂, a (C₃-C₈)cyclo(C₁ -C₈) alkoxycarbonyl, --OCOCH₂ C₆ H₅ or a substitutedarylalkoxycarbonyl wherein the substituent is a halogen, a nitro group,an amino group or a methoxyl group; or R¹ and R² taken together form,with the nitrogen to which they are attached, a 5 to 7 membered ring;and L¹ and L² are, independently, a linear or branched chain alkyl ofthe formula --(C_(n) H_(2n))-- wherein n is 1 to 8; a cycloalkyl of 3 to8 carbon atoms, or an interphenylene.
 7. A method of enhancing anantigen-specific IgG immune response to a polysaccharide comprisingadministering to a mammal in need of such production an amount of saidpolysaccharide and a compound of formula I: ##STR6## wherein: A is agroup of the formula --PO₃ H, --SO₃ H, --OPO--(OH)₂ --OSO₂ OH, or --SH,or pharmaceutically acceptable salt thereof or physiologicallyhydrolyzable derivative thereof, or disulfide thereof when A is --SH,R₁is H, a linear or branched lower alkyl, an arylalkyl or an alkenyl, R₂is H, a linear or branched lower alllyl, an alkenyl, an arylalkyl, anacyl, a carbonate ester, an alkyloxy carbonyl, a cycloalkoxycarbonyl, anunsubstituted arylalkoxycarbonyl or a substituted arylalkoxycarbonyl, orR¹ and R² taken together form, with the nitrogen to which they areattached, a 5 to 7 membered ring, and L¹ and L² are, independently, ahydrocarbon linking group, a cycloalkyl, or an interphenylene,sufficient to enhance said immune response.
 8. The method of claim 7wherein ##STR7## wherein: A is a group of the formula --PO₃ H, --SO₃ H,--OPO--(OH)₂, --OSO₂ OH, or --SH, or pharmaceutically acceptable saltthereof or physiologically hydrolyzable (C₁ -C₄)alkyl or arylalkyl esterthereof, or disulfide thereof when A is --SH;R₁ is H, a linear orbranched (C₁ to C₆) alkyl, a substituted or unsubstituted phenyl(C₁ -C₄)alkyl, or a (C₂ -C₆) alkenyl; R₂ is H, a linear or branched (C₁ to C₆)alkyl, a (C₂ -C₆) alkenyl, a substituted or unsubstituted phenyl (C₁-C₄) alkyl, an acetyl, benzoyl or arylsulfonyl, a (C₁ -C₇)alkoxycarbonyl, --OCOCH₂ CH═CH₂, a (C₃ -C₈)cyclo(C₁ -C₈) alkoxycarbonyl,--OCOCH₂ C₆ H₅ or a substituted arylalkoxycarbonyl wherein thesubstituent is a halogen, a nitro group, an amino group or a methoxylgroup; or R¹ and R² taken together form, with the nitrogen to which theyare attached, a 5 to 7 membered ring; and L¹ and L² are, independently,a linear or branched chain alkyl of the formula --(C_(n) H_(2n))--wherein is 1 to 8; a cycloalkyl of 3 to 8 carbon atoms, or aninterphenylene.
 9. The method as in one of claims 7 and 8, wherein thepolysaccharide is conjugated to a protein.
 10. The method as in one ofclaims 1, 2, 5 and 6, wherein one or more of said antigens is conjugatedto a protein.
 11. The vaccine as in one of claims 3 and 4, wherein oneof said antigens is a polysaccharide.